Significance: Chloroplast biogenesis is a fundamental process occurring during seedling ontogenesis and leading to plant autotrophy. Which membrane components sterically organize the light-triggered transition of etioplast prolamellar bodies (PLBs) into chloroplast thylakoids, and thus mediate cubic–lamellar transformation, is poorly understood. Here, we used combined two- and three-dimensional electron microscopy, spectroscopy, and biochemical methods to determine the role of CURT1 proteins in the formation of etioplast cubic membranes and their transformation to photosynthetically active chloroplast thylakoids. CURT1 proteins were previously recognized as significant contributors to thylakoid membrane folding. We found that CURT1 proteins are integral proteins of etioplast membranes and act as factors modulating PLBs and prothylakoid nanomorphology. They are also required for concerted thylakoid maturation under de-etiolation.

Photosynthetic organelles offer attractive features for engineering small molecule bioproduction by their ability to convert solar energy into chemical energy required for metabolism. The possibility to couple biochemical production directly to photosynthetic assimilation as a source of energy and substrates has intrigued metabolic engineers. Specifically, the chemical diversity found in plants often relies on cytochrome P450-mediated hydroxylations that depend on reductant supply for catalysis and which often lead to metabolic bottlenecks for heterologous production of complex molecules. By directing P450 enzymes to plant chloroplasts one can elegantly deal with such redox prerequisites. In this study, we explore the capacity of the plant photosynthetic machinery to drive P450-dependent formation of the indigo precursor indoxyl-β-D-glucoside (indican) by targeting an engineered indican biosynthetic pathway to tobacco (Nicotiana benthamiana) chloroplasts. We show that both native and engineered variants belonging to the human CYP2 family are catalytically active in chloroplasts when driven by photosynthetic reducing power and optimize construct designs to improve productivity. However, while increasing supply of tryptophan leads to an increase in indole accumulation, it does not improve indican productivity, suggesting that P450 activity limits overall productivity. Co-expression of different redox partners also does not improve productivity, indicating that supply of reducing power is not a bottleneck. Finally, in vitro kinetic measurements showed that the different redox partners were efficiently reduced by photosystem I but plant ferredoxin provided the highest light-dependent P450 activity. This study demonstrates the inherent ability of photosynthesis to support P450-dependent metabolic pathways. Plants and photosynthetic microbes are therefore uniquely suited for engineering P450-dependent metabolic pathways regardless of enzyme origin. Our findings have implications for metabolic engineering in photosynthetic hosts for production of high-value chemicals or drug metabolites for pharmacological studies.

Betalains are water-soluble pigments with two major classes: red-violet betacyanins and yellow-orange betaxanthins. These pigments are increasingly being sought after as natural replacements for synthetic pigments in the food industry. Traditionally, betalains are extracted from cultivated plants. But due to low concentrations of native pigments, the process is inherently inefficient. Now, an increase in consumer demand calls for the development of scalable and sustainable betalain production routes. To address this challenge, we introduced a heterologous pathway for the production of betaxanthins into cyanobacteria. The pathway consists of an engineered variant of the cytochrome P450 CYP76AD1 (W13L, F309L) and the l-DOPA 4,5-dioxygenase DODA1 from Beta vulgaris (beet). Introduction of the two-enzyme betaxanthin pathway in Synechocystis sp. PCC 6803 did not result in detectable betaxanthins. Subsequent metabolic adjustments to the shikimate pathway, using a feedback resistant AroG^fbr from E. coli, led to an overaccumulation of the aromatic amino acids phenylalanine, tryptophan, and tyrosine, and the production of low levels of phenylalanine-betaxanthin. Optimization of the cultivation conditions (i.e., growth in nutrient-rich medium and CO₂-enriched air) increased titers approximately 165 times and led to the production of phenylalanine-betaxanthin with a final titer of 18.2 ± 5.1 mg L^-1. Our work establishes a microbial system for photoautotrophic betaxanthin pigment production without the need for exogenous amino acid supplementation.

Betalains are water-soluble pigments with two major classes: red-violet betacyanins and yellow-orange betaxanthins. These pigments are increasingly being sought after as natural replacements for synthetic pigments in the food industry. Traditionally, betalains are extracted from cultivated plants but, due to low native pigments, the process is inherently inefficient. Now, as consumer demand increases, scalable and sustainable production routes are needed. To address this challenge, we engineered a heterologous pathway for the production of betaxanthins into cyanobacteria. The pathway consists of the cytochrome P450 CYP76AD1 and the L-DOPA 4,5-dioxygenase DODA1 from Beta vulgaris (beet). Introduction of the two-enzyme betaxanthin pathway in Synechocystis sp. PCC 6803 did not result in detectable betaxanthins. Subsequent metabolic adjustments to the shikimate pathway, using a feedback resistant AroG ^fbr from E. coli , led to an overaccumulation of the aromatic amino acids phenylalanine, tryptophan, and tyrosine, and the production of 0.11 mg L ^-1 phenylalanine-betaxanthin. Optimization of the cultivation conditions (i.e., growth in nutrient-rich medium and CO ₂ -enriched air) increased titers almost 150 times and led to the production of phenylalanine-betaxanthin with a final titer of 15.3 mg L ⁻¹ . Our work establishes a microbial system for photoautotrophic betaxanthin pigment production without the need for exogenous amino acid supplementation.

The phenylpropanoid pathway is one of the main carbon sinks in plants, channeling phenylalanine towards thousands of products including monolignols, stilbenes, flavonoids and volatile compounds. The enzymatic steps involved in many of these pathways are well characterized, however the physical organization of these enzymes within the plant cell remains unknown Proximity-dependent labeling allows untargeted determination of protein interactions in vivo , and therefore stands as an attractive alternative to targeted binary assays for determining global protein-protein interaction networks. In this study, we show a TurboID-based proximity labeling system developed to study protein interaction networks of the core phenylpropanoid pathway in petunia. Here, the endoplasmic reticulum (ER) membrane anchored cytochrome P450 cinnamic acid 4-hydroxylase (C4H, CYP73A412) from Petunia inflata was coupled to TurboID and expressed in protoplasts derived from anthocyanin-rich petunia petals. Potential interactors were isolated using streptavidin beads, digested and quantified by mass spectrometry. Among the enriched proteins, we identified multiple soluble enzymes from the late anthocyanin pathway, other CYP73 isoforms, as well as additional ER membrane anchored CYPs including p -coumaric acid 3-hydroxylase (C3’H, CYP98A2). Our results suggest that CYP73A412 co-localizes with enzymes from the phenylpropanoid- and downstream anthocyanin pathways, supporting the idea that CYP73s may serve as ER anchoring points for these metabolic pathways. Moreover, this study demonstrates the feasibility of using protoplasts to perform global mapping of protein network for enzymes in their native cellular environment.

Photosynthetic organisms such as cyanobacteria have the potential for the sustainable production of complex organic molecules due to their ability to use light as an energy source to fix CO₂ and assimilate inorganic nutrients. Over the past decade, large efforts have been put into the metabolic engineering of cyanobacteria to produce various compounds such as alcohols, isoprenoids, biopolymers, and recombinant proteins. Forskolin is a structurally complex labdane-type diterpenoid with eight chiral carbon atoms and is naturally produced in the root cork of the plant Plectranthus barbatus. Forskolin is a potent cAMP activator indicated as a pharmaceutical for a variety of diseases. In the plant, forskolin biosynthesis from geranylgeranyl diphosphate involves six enzymes: two terpene synthases, three cytochrome P450s, and a single acetyltransferase. In this work, we express all six biosynthetic genes from Plectranthus barbatus in Synechocystis sp. PCC. 6803 and demonstrate heterologous production of this complex diterpenoid in a phototroph cyanobacterium. Forskolin titers reached 25.0 ± 4.4 µg/L and the forskolin was entirely secreted into the media. The forskolin-producing Synechocystis strain and empty vector control were cultivated in a photobioreactor for 8 days. Both strains showed similar chlorophyll a contents, and the forskolin-producing strain reached a slightly higher OD₇₃₀ than the control. Forskolin began accumulating in the supernatant after 4 days and increased over time. These results indicate that forskolin production did not negatively impact cell growth.